Decapeptides produced from bioadhesive polyphenolic proteins

ABSTRACT

Methods are described for the preparation and isolation of novel decapeptides of the formula: ##STR1## wherein each X is independently selected from the group comprising hydroxyl and hydrogen, wherein each R is independently selected from the group comprising hydrogen and methyl, from bioadhesive polyphenolic proteins which comprise: ##STR2## wherein n is a whole number from about 60 to about 100, wherein each X is independently selected from the group comprising hydroxyl and hydrogen, and wherein each R is independently sselected from the group comprising hydrogen and methyl. 
     Such decapeptides may be used to construct large polyphenolic molecules comprising from about 1 to about 1000 decapeptide repeating units and wherein the linking group is selected from the group comprising amino acid, oligopeptide and bifunctional spacer.

This application is a division of application Ser. No. 820,143, filedJan. 21, 1986 and now U.S. Pat. No. 4,687,740 which was a division ofapplication Ser. No. 587,132, filed Mar. 7, 1984 and now U.S. Pat. No.4,585,585.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to bioadhesive polyphenolic proteinsderived from several species of the mussel genus Mytilus. Thesepolyphenolic proteins, which contain a sequence of repeatingdecapeptides, exhibit unusually superior adhesive capabilities toward avariety of surfaces including surfaces submerged in water.

2. Description of the Prior Art

Adhesives well known in the art are generally applied to dry surfaces inorder to effect a strong bond. The vast majority of adhesives bind drysurfaces more strongly than the same surfaces when wet. For example,resorcinol-formaldehyde polymers which are useful in making waterproof,boil-proof plywood and particle board cannot be applied to surfacesunderwater due to the dispersive effect of water on the monomers(resorcinol and formaldehyde). For these compositions to form a strongbond, the monomers must be mixed, set and cured at about 10 to about 50%relative humidity at temperatures equal to or exceeding about 20° C.Thus, present adhesive technology is stymied by the presence of water onsubstrates; water competes with the adhesive for surface area on whichto bind. In addition, for many adhesives, water tends to hydrolyze orplasticize the adhesive.

Methods for the isolation of polyphenolic proteins from the mussel genusMytilus are known in the art, and are described in the article of Waiteand Tanzer, Science 212, 1038 (May 29, 1981). Heretofore, however, nomethod for the preparation and isolation of decapeptides frompolyphenolic proteins have been known.

Thus, it is an object of this invention to provide a method for thepreparation of repeating decapeptides from said bioadhesive proteins.

It is yet another object of the present invention to providedecapeptides for preparing larger bioadhesive molecules useful inbinding surfaces in the presence of water.

SUMMARY OF THE INVENTION

The foregoing and other objects, advantages, and features of thisinvention may be achieved by the use of bioadhesive polyphenolicproteins which comprise: ##STR3## wherein n is a whole number from about60 to about 100, wherein each X is independently selected from the groupcomprising hydroxyl and hydrogen and wherein each R is independentlyselected from the group comprising hydrogen and methyl.

Digestion of the polyphenolic proteins in trypsin in the presence of aneutral or slightly basic buffer results in decapeptides of the formula:##STR4## wherein each X is independently selected from the groupcomprising hydroxyl and hydrogen and wherein each R is independentlyselected from the group comprising hydrogen and methyl.

Such decapeptides may be used as "building blocks" in the constructionof larger polyphenolic molecules possessing the adhesive capabilities ofthe native bioadhesive protein, comprising from about 1 to about 1000decapeptide repeating units and wherein the linking group comprisesamino acid, oligopeptide or bifunctional spacer.

DETAILED DESCRIPTION OF THE INVENTION

Bioadhesive polyphenolic proteins are characterized by a very lowaqueous dispersive effect probably due, at least in part, to the highamounts of hydroxyproline (HYP) and 3,4-dihydroxyphenylalanine (DOPA)present in the proteins. Such bioadhesive proteins are furthercharacterized by their low solubility at neutral or slightly basic pH.In addition, such proteins behave as polymers and adhere to teeth andbone surfaces as well as wood, glass, iron, steel, slate, and so forthunder water (at about 300 to about 1000 pounds per square inch). Suchbonds appear to be durable in the presence of water for years.

These bioadhesive proteins may be isolated and purified from dissectedphenol glands of mussels. Because bioadhesive proteins exhibit a ratherlow solubility at neutral pH, these proteins may be purified initiallyby extracting impurities, including extraneous proteins, with largeamounts of a neutral or slighly basic salt buffer followed by gentlecentrifugation. The neutral or slightly basic salt buffer containsvarious protease inhibitors to prevent premature degradation of thebioadhesive proteins as well as cyanide which prevents enzymaticoxidation of the DOPA residues prevalent in the bioadhesive proteins. Itis important to use a gentle first centrifugation to prevent theirreversible coalescence of insoluble proteins, which include thebioadhesive proteins. After centifugation, the insoluble materials arere-extracted with an acidic solution, most preferably acetic acid, inwhich the polyphenolic proteins are very soluble. It should be notedthat a high yield purification of the bioadhesive proteins is difficultdue to the extensive adsorption of the proteins to surfaces.

Bioadhesive proteins may be purified by a combination of ion exchangeand gel filtration on low surface-energy chromatographic media. Ionexchange on sulfonylpropyl-Sephadex provides a most effectivepurification step. Gel filtration of bioadhesive proteins using avariety of chromatographic materials and buffers generally result in avery low or negligible yield. Yield can be improved on Sephadex if anelution buffer with a low pH (in the range of about 2 to about 4) and acationic detergent are used. Although recovery from phenyl-Sepharose 4Bis excellent, this material provides a limited fractionation range andis generally not preferred for purifying the bioadhesive proteins. Theapparent molecular weight of the bioadhesive proteins, as determined bypolyacrylamide gel electrophoresis in the presence of cetylpyridiniumbromide, is estimated to be about 120,000 to about 140,000.

When the bioadhesive proteins are treated with clostridial collagenase,the molecular weight is reduced to between about 110,000 and about130,000. Collagenase digestion may be performed in a salt buffersolution with collagenases of varying purity. It is preferred to usecollagenase of high purity in a neutral or slightly basic buffer, mostpreferably a borate-salt buffer. The resultant collagenase-resistantfragments contain most of the HYP and DOPA of the original bioadhesiveproteins.

Collagenase-resistant fragments are rapidly degraded by trypsin intodecapeptides, the repeating unit in the native protein. Trypsindegradation of the collagenase-resistant fragments may be performed in asalt buffer at neutral or slightly basic pH. Again, the use of aborate-salt buffer is especially preferred. At the termination of thetrypsin digestion, the decapeptides may be purified by gel filtrationdialysis, or a combination of known purification techniques.

Alternatively, trypsin digestion may be performed on isolatedbioadhesive proteins, producing the same decapeptides, without firsttreating the bioadhesive proteins with clostridial collagenase. Trypsindegradation is again performed in a salt buffer at neutral or slightlybasic pH, with the use of a borate-salt buffer being especiallypreferred. At the termination of the trypsin digestion, the decapeptidesmay be purified from the collagenase-labile fragments by gel filtrationdialysis, or by a combination of known purification techniques.

These decapeptides comprise: ##STR5## wherein each X is independentlyselected from the group comprising hydroxyl and hydrogen and whereineach R is independently selected from the group comprising hydrogen andmethyl.

These decapeptides, which are principally responsible for thebioadhesive properties of the bioadhesive proteins, may be repeated from60 to about 100 times in bioadhesive polyphenolic proteins isolated fromthe marine mussel. Because of the prevalence of hydroxyl-substitutedphenyl groups in the decapeptides, the parent bioadhesive protein isoften referred to as a "polyphenolic" protein.

It is believed that the large amounts of hydroxyproline (HYP) and3,4-dihydroxylphenylalanine (DOPA) as well as the numerous hydroxylgroups in the polyphenolic proteins are largely responsible for thenon-dispersive properties of the protein.

It is possible to construct large polyphenolic molecules possessing theadhesive capabilites of the naturally occurring bioadhesive proteins,comprising from about 1 to about 1000 decapeptide repeating units andwherein the linking group comprises amino acid, oligopeptide andbifunctional spacer. Known bifunctional compounds may be used to inducethe polyermization of the decapeptides. Virtually any bifunctionalcompound in which both functionalities react with or become ionicallyassociated with hydroxyl groups, amine groups or carboxyl groups may beused. While aqueous salt buffers at neutral or slightly basic pH may beused as a reaction medium, the use of organic solvents may likewise beused. The reaction of the decapeptides and bifunctional linking group iscontinued at a temperature, and for a time period necessary tosubstantially complete the polyermization.

Classic methods of protein synthesis may also be used to linkdecapeptides to form larger polyphenolic molecules. Methods contemplatedherein include well-known blocking, activating, linking, and deblockingsequences of peptide synthesis. While no linking group is necessary insuch syntheses, linking groups of amino acids and oligopeptides may beused.

Amino acids which may be used as linking groups in the construction oflarge polyphenolic molecules comprise any of the naturally occurringL-amino acids, as well as other amino acids, such as ornithine,homocysteine, citrulline, 3-aminotyrosine, and the like.

Oligopeptides which may be used as linking groups comprise any di-,tri-, tetra- or penta-peptides and higher peptides thay may be readilysynthesized or available from commercial sources. Examples, ofoligopeptides include (ala-cys-ala), (ala-lys)₃, (ala-lys-pro)₄,(pro-hyp-gly)₅, and the like.

Bifunctional spacers which may be used as linking groups comprisealiphatic or aromatic dialdehydes, imido esters, diisocyanates, aryl andalkyl dihalides, dimaleimides, and the like. The dialdehydes may be ofthe type: OHC--R--CHO, wherein R is selected from the group comprisinglower alkyl, aryl or substituted aryl. Examples of suitable dialdehydesinclude glutaraldehyde, malonaldehyde, glyoxal, 1,4-butanedialdehyde,and the like. Useful imido esters comprise: ##STR6## wherein R, R' andR" are independently selected from the group comprising lower alkyl,aryl or substituted aryl. Examples of suitable imido esters includedimethyl malonimidate, dimethyl suberimidate and dimethyl adipimidate.

Useful diisocyanates comprise: O═C═N--R--C═N═O, wherein R is selectedfrom the group comprising lower alkyl, aryl, and substituted aryl.Examples of suitable diisocyanates include pentamethylene diisocyanate,hexamethylene diisocyanate, heptamethylene diisocyanate, andtoluene-2,4-diiosocyanate. Useful aryl dihalides comprise: ##STR7##wherein X and X' are independently selected from the group comprising F,Cl, Br, and I and R₁, R₂, R₃, and R₄ are selected independently from thegroup comprising lower alkyl, aryl or substituted aryl. Examples ofsuitable aryl dihalides include p-dibromobenzene, o-bromoiodobenzene,2,4-dibromotoluene and the like. Useful alkyl dihalides comprise:X--R--X', wherein X and X' may be independently selected from the groupcomprising F, Cl, Br and I; and R may be alkyl or substituted alkyl.Examples of alkyl dihalides include 1,2-dibromoethane,1,3-dibromopropane, methylene bromide, methylene iodide and the like.

Dimaleimides which may be used comprise: ##STR8## wherein R and R₁ areindependently selected from the group comprising hydrogen, lower alkylor aryl. Examples of suitable dimaleimides includebis(N-maleimidomethyl) ether and the like.

EXAMPLE 1 Isolation and Purification of Bioadhesive PolyphenolicProteins

Prior to the isolation of bioadhesive polyphenolic proteins, musselphenol glands were isolated and extracted, generally in accordance withthe method described by Waite and Tanzer, Science 212, 1038 (May 29,1981). The shells of fresh live mussels (Mytilus edulis) were opened bycutting the anterior and posterior adductor muscles with a scalpel. Theanimals were then killed by cardiac puncture and the foot from eachanimal was amputated at the base and placed on ice. Mussel feet in lotsof 30 were transferred to clean glass plates (20×20×0.2 cm) and frozenover dry ice. The phenol gland which can readily be located near thefoot tip was dissected from each foot after stripping off the pigmentedepithelium. The phenol glands from 450 mussel feet were suspended in 750ml. buffer containing 1 M NaCl, 0.05 M tris (pH 7.5), 1 mMphenylmethylsulfonylfluoride, 10 mN N-ethylmaleimide, 0.025 Methylenediamine tetraacetic acid and 1 mM potassium cyanide andhomogenized in ground glass tissue grinders at 4° C. The homogenateswere lightly centrifuged for 5 min. at about 2500 X g and thesupernatants were discarded. The pellets were resuspended in cold 0.8 Macetic acid (150 ml) and rehomogenized briefly with tissue grinders. Thesecond homogenate was centrifuged for one hour at about 40,000 X g andabout 4° C. The supernatant was rich in polyphenolic proteins.

Polyphenolic proteins were isolated in two steps. First, the acetic acidsupernatant was dialyzed against a large volume of 0.8 M acetic acid.The nondialyzable fraction was adjusted with guanidine-HCl to aconductance of 30 mMHO and a final composition of 5.5% guanidine-HCl,0.8 M acetic acid and 0.001% (v/v) Triton X-100 (Buffer A). This wasapplied to a column of sulfonylpropyl-Sephadex C-50 (25×1 cm)pre-equilibrated at about 20° C. with a buffer of the same composition.Polyphenolic proteins were eluted from the column at a bufferconductance from about 40 to about 60 mMHO by using a linear gradientproduced by mixing the first buffer (A) with 20% guanidinehydrochloride, 0.8 M acetic acid and 0.001% Triton X-100. Yields ofpolyphenolic proteins were substantially better when chromatographed onsulfonylpropyl-Sephadex possessing manufacturer's four-digit batchnumbers than when chromatographed on sulfonylpropyl-Sephadex withmanufacturer's five-digit designations. Following ion exchange, peakfractions of polyphenolic proteins were dialyzed against 0.8 M aceticacid to remove detergent and guanidine-HCl. Further purification wasachieved on columns (both 62×2 cm) of Sephadex G-200 andPhenyl-Sepharose 4B eluted with 0.3 M ammonium acetate (pH 4.0) and0.01% cetylpyridinium bromide, and 3% guanidine in 0.8 M acetic acid,respectively. Numerous other buffers and chromatographic media weretested, but recoveries of polyphenolic proteins were, in most instances,too low to merit further description.

EXAMPLE 2 Trypsin Digestion of Polyphenolic Proteins

For trypsin digestion, 5 mg of the polyphenolic proteins were dialyzedagainst 0.01 M sodium borate (pH 8.5) with 3 M urea and 0.01 mM CaCl₂.Trypsin was added at an enzyme to substrate ratio of about 1 to 100, andthe reaction was stirred under oxygen-free nitrogen at 25° C. for 24hours. At the end of this period, the reaction was terminated by addinga few drops of glacial acetic acid (to pH 4) and flash evaporated toabout 0.5 mL. Sample volume was adjusted to 1 mL with 0.2 M acetic acidand centrifuged to remove insoluble material. The supernatant wasapplied to a column of Sephadex LH 60 (80×1.5 cm) eluted with 0.2 Macetic acid. Fractions were assayed for ninhydrin-positive material andDOPA. The material containing both DOPA and amines was pooled, flashevaporated and resuspended in 0.2M pyridine acetate (pH 3.1). Thissample was applied to a Sephadex SP-25 column (20×1 cm) eluted with alinear pH gradient ranging from pH 3.1 to pH 5.0 (2.0 M pyridineacetate). Fractions were tested as described above. Again, theDOPA-rich, ninhydrin-positive peak fractions were flash evaporated andpurified on a column of Sephadex LH 20 (80×1.5 cm) eluted with 0.2 Macetic acid. This peak was flash evaporated to dryness and stored at-20° C.

Trypsin digestion of the polyphenolic proteins is extensive and rapid.Under the conditions used, the proteins completely disappeared within 5min. of the addition of trypsin as determined by gel electrophoresis.Fractionation of the tryptic digest was achieved using gel filtration toSephadex LH-60, which removes trypsin from the peptides, followed by ionexchange on Sephadex SP-25 with a pyridine acetate gradient (the lattermethod being recommended for basic aromatic peptides). About 75-80% ofthe ninhydrin-positive material eluting from SP-Sepadex can be ascribedto the DOPA-containing peak. Moreover, nearly 95% of the DOPA and 80% ofthe proteins, originally applied to SP-Sephadex are recovered is themajor DOPA rich peak. This material was further purified by passagethrough Sephadex LH-20. The tryptic decapeptides resemble thepolyphenolic proteins in containing the same group of amino acids thatpredominate in the latter, namely HYP, THR, SER, PRO, ALA, DOPA, TYR,and LYS. In the tryptic decapeptides, however, DOPA and HYP aresignificantly enriched, whereas LYS, PRO and TYR are reduced. Thetryptic decapeptides were homogeneous on 12% acrylamide gels in a 3 Murea and 5% acetic acid but were visualized by DOPA staining since theycould not be fixed for protein staining. Molecular weight of thedecapeptides in cetylpyridinium bromide gel electrophoresis wasestimated to be about 6500. Peptide homogeneity was also suggested bythin layer chromatography on cellulose in 1.5% formic acid and thinlayer electrophoresis in 5% acetic acid. Since borate strongly complexesDOPA at pH 7-9, it has the property of introducing additional negativecharges into the decapeptides. Heterogeneity of the decapeptides (2spots) in borate suggests variation in the degree of TYR to DOPAconversion.

Sequenator analysis (35 cycles) of the tryptic decapeptides revealed itto be a mixture of decapeptides (molecular weight of about 1400)comprising: ##STR9## wherein each X is independently selected from thegroup comprising hydrogen and hydroxyl and wherein each R isindependently selected from the group comprising hydrogen and methyl.

EXAMPLE 3 Enzymatic Digestion of Polyphenolic Proteins with ClostridialCollagenase

As described above, polyphenolic protein may be enzymatically digestedto prepare novel decapeptides by the use of trypsin alone. In addition,purified polyphenolic proteins may be enzymatically digested to preparenovel decapeptides with the successive use of two proteases--clostridialcollagenase and trypsin. This example describes the degradation of thepolyphenolic proteins with clostridial collagenase.

Collagenase used was of high specific activity (7×10⁻¹¹ moles leucinereleased/min/mg) and had no detectable endopeptidase activity withcasein as a substrate. Polyphenolic proteins (1 mg) were dialyzedagainst 0.1 M sodium borate pH 8.0 with 0.01 mM CaCl₂ under nitrogen.Collagenase was added at an enzyme to substrate ratio of 1:250. Thereaction mixture was incubated at 35° C. under continuous stirring and50 microliter aliquots were periodically removed for electrophoreticanalysis. Collagenase activity was terminated by lowering aliquot pH to4.0 with acetic acid.

Collagenase treatment of polyphenolic proteins results in only limiteddegradation. Only about 8% of protein was attacked, leaving entirelyintact a fragment with M_(r) =120,000. This collagenase-resistantfragment has an amino acid composition similar to that of the originalprotein but is noticeably reduced in glycine and proline. Clostridialcollagenase selectively cleaves proline-glycine linkages in thepolyphenolic proteins.

EXAMPLE 4 Polymerization of Bioadhesive Decapeptide Using a BifunctionalLinker

Decapeptides may be polymerized using a glutaraldehyde linking group asfollows: Decapeptides (4 mg) prepared from the isolated polyphenolicprotein by trypsin degradation, as described above in Example 2, aremixed in sodium acetate (1 ml, 0.2M, pH 7). Aqueous glutaraldehyde (1ml, 25% w/v) is added in a dropwise fashion. The mixture is stirredvigorously for about 90 minutes at room temperature. At the end of thistime, the mixture is dialzyed against 1000 volumes of distilled water atabout 4° C. for about six hours. The nondialyzable fraction isfreeze-dried. The polymerization is confirmed by gel electrophoresis ofthe product in the presence of cetylpyridinium bromide.

While the foregoing is intended to illustrate methods for the isolationof bioadhesive polyphenolic proteins and the preparation of decapeptidestherefrom as well as the polymerization of the decapeptides, theseexamples are not intended nor should they be construed as a limitationon the invention. As one skilled in the art would understand, manyvariations and modifications may be made in these processes andcompositions that fall within the spirit and scope of this invention.

I claim:
 1. A composition prepared by a method which comprises the stepsof:(a) isolating bioadhesive proteins from mussel phenol glands; (b)enzymatically digesting said bioadhesive protein; and (c) recoveringsaid decapeptides.
 2. A composition prepared by a method which comprisesthe steps of:(a) isolating from marine mussels bioadhesive polyphenolicproteins which comprise: ##STR10## wherein n is a whole number fromabout 60 to about 100, wherein each X is independently selected from thegroup comprising hydroxyl and hydrogen, and wherein each R isindependently selected from the group comprising hydrogen and methyl;(b) digesting said bioadhesive proteins with clostridial collagenase;(c) digesting the resultant collagenase-resistant fragment with trypsin;and (d) recovering said decapeptides.
 3. A method for the preparation ofpolyphenolic molecules consisting of from about 1 to about 1000repeating decapeptide units, which method comprises the step of reactingdecapeptides of the formula: ##STR11## wherein each X is independentlyselected from the group comprising hydroxyl and hydrogen, and whereineach R is independently selected from the group comprising hydrogen andmethyl, with a linking group which is selected from the group comprisingamino acid, oligopeptide, and bifunctional spacer, in a neutral orslightly basic buffer, at a temperature, and for a period of time untilthe reaction is substantially complete.
 4. Polyphenolic molecules whichcomprise from about 1 to about 1000 repeating units of the formula:##STR12## wherein each X is independently selected from the groupconsisting of hydroxyl and hydrogen and wherein each R is independentlyselected from the group consisting of hydrogen and methyl, and whereinthe repeating units are linked.